Somatic hypermutation of variable genes, which encode a portion of immunoglobulin molecules, occurs at a frequency that is a million times greater than mutation in other genes. The molecular mechanism that introduces these mutations is unknown. Evidence points to a process that involves DNA repair events at sites of targeted strand breaks. In vertebrate cells, there are many recently identified DNA polymerases that inaccurately copy templates. One or more of these are potential candidates for enzymes that introduce base changes during hypermutation. We are studying the roles of DNA polymerases zeta, eta, and iota in the mechanism. (a) Polymerase zeta creates errors at a frequency of 5 X 10-4/bp when copying undamaged DNA. In collaboration with R. Wood, we disrupted the catalytic subunit of the polymerase and made gene-deficient mice. However, the mice died during mid-gestation, suggesting that the enzyme is critical for embryonic development. (b) Polymerase eta synthesizes errors at a frequency of 3 X 10-2/bp on non-damaged DNA. The enzyme is defective in people with xeroderma pigmentosum variant disease. We sequenced variable genes from three patients and found that their frequency of hypermutation was normal, but the types of base changes were different. Polymerase eta-deficient clones had a three-fold decrease in the proportion of mutations at A and T with a concomitant rise of mutations at G and C. It is notable that this shift in mutation pattern is consistent with the specificity of the polymerase when copying non-damaged DNA in vitro. This finding implies that polymerase eta is an A-T mutator in hypermutation, and another polymerase acts at G and C nucleotides. (c) Polymerase iota makes errors at an overall frequency of 3 X 10-1/bp on undamaged templates. In collaboration with R. Woodgate, we have studied the specificity of polymerase iota on DNA substrates that might be formed during hypermutation. The fidelities of the polymerase are 10-fold lower when it fills a template at a DNA terminus compared to when it fills a longer template. Since mutations occur in variable genes near strand breaks, the polymerase may well function as a mutator during hypermutation.